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Mechanics-Guided Embryonic Patterning of Neuroectoderm Tissue from Human Pluripotent Stem Cells

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Mechanics-Guided Embryonic Patterning of Neuroectoderm Tissue from Human Pluripotent Stem Cells ARTICLES https://doi.org/10.1038/s41563-018-0082-9 Mechanics-guided embryonic patterning of neuroectoderm tissue from human pluripotent stem cells Xufeng Xue1,9, Yubing Sun1,2,9*, Agnes M. Resto-Irizarry1, Ye Yuan3, Koh Meng Aw Yong1, Yi Zheng1, Shinuo Weng 1, Yue Shao 1, Yimin Chai4, Lorenz Studer5,6 and Jianping Fu 1,7,8* Classic embryological studies have successfully applied genetics and cell biology principles to understand embryonic develop- ment. However, it remains unresolved how mechanics, as an integral driver of development, is involved in controlling tissue-scale cell fate patterning. Here we report a micropatterned human pluripotent stem (hPS)-cell-based neuroectoderm developmental model, in which pre-patterned geometrical confinement induces emergent patterning of neuroepithelial and neural plate border cells, mimicking neuroectoderm regionalization during early neurulation in vivo. In this hPS-cell-based neuroectoderm pattern- ing model, two tissue-scale morphogenetic signals—cell shape and cytoskeletal contractile force—instruct neuroepithelial/ neural plate border patterning via BMP-SMAD signalling. We further show that ectopic mechanical activation and exogenous BMP signalling modulation are sufficient to perturb neuroepithelial/neural plate border patterning. This study provides a use- ful microengineered, hPS-cell-based model with which to understand the biomechanical principles that guide neuroectoderm patterning and hence to study neural development and disease. ne of the enduring mysteries of biology is tissue morpho- on glass coverslips (Fig. 1a and Supplementary Fig. 1). H1 human genesis and patterning, where embryonic cells act in a embryonic stem (hES) cells were plated as single cells at 20,000 coordinated fashion to shape the body plan of multicellu- cells cm−2 on adhesive islands to establish micropatterned colonies O 1–5 lar animals . As a highly conserved developmental event crucial with a defined circular shape and size. A differentiation medium for the nervous system formation, neural induction, for example, supplemented with the dual SMAD inhibitors, SB 431542 (SB, leads to differentiation of the ectoderm into a patterned tissue, con- TGF-β inhibitor; 10 µ M) and LDN 193189 (LDN, BMP4 inhibitor; taining the neuroectoderm (neural plate, or NP) and the epidermal 500 nM), was applied for neural induction22 (Supplementary Fig. 1; ectoderm separated by the neural plate border (NPB) (Fig. 1a)6,7. see Methods). The β -catenin stabilizer CHIR 99021 (CHIR, 3 µ M), Classic embryological studies of neural induction have unravelled a WNT activator, was also supplemented in culture (Supplementary the importance of graded developmental signalling mediated by Fig. 1). CHIR promotes NPB cell specification under the neural diffusible signals such as bone morphogenetic proteins (BMPs) induction condition established by the dual SMAD inhibitors23,24. (Fig. 1a)8–10. However, neural induction, like any tissue-scale mor- Although cells distributed uniformly on adhesive islands 24 h phogenetic event, occurs within the milieu of biophysical determi- after initial cell plating, neural induction resulted in differentiat- nants, including changes in shape, number, position and force of ing cells gradually accumulating in the colony central area, lead- cells7,11. Yet it remains undetermined how these tissue-scale mor- ing to a significantly greater cell density at the colony centre than phogenetic changes work in concert with classic developmental at the periphery (Fig. 1b and Supplementary Fig. 1). Cell density signaling events mediated by diffusible signals for proper cell fate was further analysed using DAPI fluorescence intensity. The full patterning during neural induction. width at half maximum (FWHM) for spatial distributions of DAPI hPS cells, which reside in a developmental state similar to the intensity decreased continuously from 336 µ m at day 1 to 240 µ m pluripotent epiblast12,13, have been successfully used for the devel- at day 9 (Supplementary Fig. 1). Confocal images further showed opment of self-organized models of human embryonic develop- that micropatterned colonies at day 7 remained as a monolayer. ment14–21. To date, however, no neural induction models exist Strikingly, quantitation of colony thickness and nucleus shape that leverage hPS cells and their innate self-organizing properties revealed that at this point, cells exhibited a gradual change of cell to study neuroectoderm patterning. Here, we sought to develop shape from a columnar phenotype with columnar nuclei at the col- micropatterned hPS-cell colonies on two-dimensional substrates to ony centre to a cuboidal morphology with rounded nuclei at the model neural induction. Microcontact printing was utilized to gen- colony periphery (Supplementary Fig. 1), consistent with character- erate vitronectin-coated, circular adhesive islands with a diameter istic neuroectoderm thickening during neural induction in vivo6,11. of 400 µ m on flat poly-dimethylsiloxane (PDMS) surfaces coated Importantly, Pax6+ neuroepithelial (NE) cells, the neural progenitor 1Department of Mechanical Engineering, University of Michigan, Ann Arbor, MI, USA. 2Department of Mechanical and Industrial Engineering, University of Massachusetts, Amherst, MA, USA. 3School of the Gifted Young, University of Science and Technology of China, Hefei, China. 4Department of Orthopedic Surgery, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai, China. 5Developmental Biology Program, Memorial Sloan- Kettering Institute, New York, NY, USA. 6Center of Stem Cell Biology, Memorial Sloan-Kettering Institute, New York, NY, USA. 7Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI, USA. 8Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI, USA. 9These authors contributed equally: Xufeng Xue, Yubing Sun *e-mail: [email protected]; [email protected] NATURE MATERIALS | VOL 17 | JULY 2018 | 633–641 | www.nature.com/naturematerials 633 © 2018 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. ARTICLES NATURE MATERIALS a b Day: 136 9 In vivo In vitro hPS cells Phase Ectoderm Mesoderm PDMS Endoderm Cover slip Neural induction Neural plate (neuroectoderm) Neural plate DAPI NPB NPB Epidermal ectoderm BMP activity Micropatterned High Low High cell colony Intensity map ncolony = 24 ncolony = 35 ncolony = 20 ncolony = 59 0–1 (arbitrary units) c PAX6 PAX3 ZIC1 MSX1 d Day: 4 678 Phase Staining image GFP Intensity map p p Intensity ma ncolony = 132 ncolony = 30 ncolony = 49 ncolony = 53 ncolony = 60 ncolony = 60 ncolony = 60 ncolony = 60 Relative intensity ma 0–1 (arbitrary units) 0–1 (arbitrary units) Fig. 1 | Self-organized neuroectoderm patterning in circular hPS cell colonies. a, Schematic of neural induction in vivo and in vitro. During neural induction in vivo, embryonic cells in the ectoderm form the neural plate (neuroectoderm). Embryonic cells at the neural plate border (NPB) separate the neuroectoderm from the epidermal ectoderm. Neural induction of circular hPS cell colonies leads to autonomously patterned neuroectoderm tissues, with neural plate cells in the colony central region and NPB cells at the colony periphery. b, Representative phase contrast and fluorescence images showing cell morphology and nuclei (stained by DAPI), respectively, on different days. White dashed lines mark the colony periphery. Experiments were repeated three times with similar results. Bottom average intensity maps show spatial distributions of DAPI intensity. The number of colonies analysed was pooled from n = 3 independent experiments. Data plotted are mean values. c, Representative immunofluorescence micrographs and average intensity maps showing colonies at day 9 stained for neuroectoderm marker Pax6 and NPB markers Pax3, Zic1 and Msx1. White dashed lines mark the colony periphery. Experiments were repeated three times with similar results. Relative intensity maps were normalized to DAPI signals. The number of colonies analysed was pooled from n = 3 independent experiments. Data plotted are mean values. d, Representative phase contrast and fluorescence images and average intensity maps from live cell assays using Sox10-EGFP hES cells. White dashed lines mark the colony periphery. White arrowheads mark GFP+ cells at the colony border. Experiments were repeated three times with similar results. The number of colonies analysed was pooled from n = 3 independent experiments. Data plotted are mean values. Scale bars in b–d, 100 µ m. in the NP, were found preferentially localized at the colony central tion is consistent with in vivo findings that ZIC1+ cells also exist in region on day 9, whereas Pax3+, Zic1+ and Msx1+ NPB cells were con- the neurogenic placode region25. Immunostaining and immunob- centrated at the colony periphery, forming a concentric ring-shaped lotting of E-cadherin and N-cadherin at day 2 and day 7 further tissue sheet consistent with neuroectoderm patterning, with proper confirmed successful neural conversion (Supplementary Fig. 2). To regionalization of NE and NPB cells (Fig. 1c and Supplementary confirm differentiation potentials of neuroectoderm tissues after Fig. 1). Interestingly, the concentric zone of Pax3+ cells at the neural induction, they were continuously cultured under a motor colony periphery exhibited a smaller zone width, as character- neuron differentiation medium26 or a neural crest differentiation ized by FWHM for spatial distribution of Pax3 intensity (92 µ
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